CN115323073A

CN115323073A - Primer probe composition and kit for detecting human cytomegalovirus nucleic acid and use method thereof

Info

Publication number: CN115323073A
Application number: CN202210161876.9A
Authority: CN
Inventors: 杨莹莹; 刘圆; 魏星
Original assignee: Beijing Huanuo Aomei Gene Medical Laboratory Co ltd
Current assignee: Beijing Huanuo Aomei Gene Medical Laboratory Co ltd
Priority date: 2022-02-22
Filing date: 2022-02-22
Publication date: 2022-11-11

Abstract

The invention provides a primer probe composition for detecting human cytomegalovirus nucleic acid, a kit and a use method thereof, belonging to the field of biological detection. The primer probe composition for detecting the human cytomegalovirus nucleic acid comprises a real-time fluorescent PCR primer and a probe, wherein the real-time fluorescent PCR primer comprises a nucleotide sequence shown in SEQ ID No. 1-2; the probe comprises a nucleotide sequence shown as SEQ ID No. 3. The kit comprises the primer probe composition. The kit realizes the detection of human cytomegalovirus nucleic acid by using real-time fluorescent signals and using a Taqman probe under the action of taqDNA polymerase after extracting nucleic acid from a collected sample by a conventional method. The kit does not have a complex nucleic acid extraction process, the fluorescent quantitative PCR process is carried out by adopting a one-step method, and the kit has the advantages of high sensitivity, short detection time, low false negative, quickness, simplicity and convenience.

Description

Primer probe composition and kit for detecting human cytomegalovirus nucleic acid and use method thereof

Technical Field

The invention belongs to the technical field of biological monitoring, and particularly relates to a primer probe composition for detecting human cytomegalovirus nucleic acid, a kit and a use method thereof.

Background

Human Cytomegalovirus (HCMV) is also known as Human herpesvirus type 5, belongs to the beta subfamily of the herpesviridae family, and is the causative agent of Cytomegalovirus inclusion body disease. The human cytomegalovirus has a circular shape, a diameter of 100-200 nm, and a genome of linear dsDNA. Human is the only natural host of HCMV, a widely transmitted human pathogen that can be naturally transmitted through saliva, urine, or lactation. It can also be transmitted by sexual contact, blood transfusion and organ transplantation.

The infection rate of HCMV in people is very high, and the HCMV antibody positive rate of adults in China reaches 90 percent. Primary infection occurs mostly under the age of 2 years, usually with occult infection as the main, and only a few have clinical symptoms. However, under certain conditions, viruses can invade multiple organs and systems to cause serious diseases. Infections are common in pregnant women, fetuses and newborns, and 80% of fetuses and newborns infected with primary HCMV during pregnancy can cause mental retardation, deformity and death; after infection of infants, serious diseases such as hepatosplenomegaly, mental retardation, choroidal optic neuritis and the like can be caused. Patients with immunodeficiency and after organ transplantation receive immunosuppressive therapy and malignant tumor therapy, serious HCMV infection is often caused, systemic diseases are often caused, and the disease death rate is high.

Therefore, the establishment of a method for rapidly and accurately detecting human cytomegalovirus infection is of great significance.

At present, virus isolation and culture are the standard of HCMV infection detection in a clinical laboratory, but the detection period is as long as 30 days, so the virus isolation and culture is not suitable for being used in the clinical laboratory. At present, many products exist in the aspect of HCMV detection in China, most of the products are products for detecting human cytomegalovirus IgG/IgM based on an immune method, and a kit for detecting the human cytomegalovirus based on a PCR technology is few, has low automation degree and high false negative.

Disclosure of Invention

The invention provides a primer probe composition for detecting human cytomegalovirus nucleic acid, a kit and a use method thereof, aiming at the difficulty of detecting the existing clinical human cytomegalovirus. When the kit is used for detecting the human cytomegalovirus nucleic acid, the sensitivity is high, the detection time is short, the false negative is low, and the kit is quick, simple and convenient.

The invention is realized by the following technical scheme:

in a first aspect, the present invention provides a primer probe composition for detecting human cytomegalovirus nucleic acid, the primer probe composition comprising real-time fluorescent PCR primers and probes;

the real-time fluorescent PCR primer comprises nucleotide sequences shown in SEQ ID No. 1-2;

the probe comprises a nucleotide sequence shown as SEQ ID No. 3;

specifically, the sequence of the primer probe composition is as follows:

further, in a preferred embodiment of the present invention, the probe is a Taqman probe, wherein a fluorescence reporter group is labeled at the 5 'end of the Taqman probe, and a fluorescence quencher group is labeled at the 3' end of the Taqman probe.

Further, the fluorescent reporter group comprises at least one of FAM, cy5, ROX, VIC, and NED; preferably, the fluorescent reporter group is FAM.

Further, the fluorescence quenching group comprises at least one of MGB-NFQ, QSY and BHQ 1; the fluorescence quenching group is MGB-NFQ.

In a second aspect, the present invention provides a kit for detecting human cytomegalovirus nucleic acids comprising: the primer probe composition described above.

Further, in a preferred embodiment of the present invention, the kit further comprises an internal reference primer and an internal reference probe specific to the conserved sequence of the human HBB gene;

preferably, the internal reference primer comprises a nucleotide sequence shown in SEQ ID No. 4-5;

preferably, the internal reference probe comprises a nucleotide sequence shown as SEQ ID No. 6;

specifically, the sequence of the primer probe composition is as follows:

HBB	nucleotide sequence	Serial number
			Forward sequence (F)	5’-TTGGACCCAGAGGTTCTTTGA-3’	SEQ ID No.4
Reverse sequence (R)	5’-GCCATGAGCCTTCACCTTAGG-3’	SEQ ID No.5
			Internal reference probe (P)	VIC-5’-TCCACTCCTGATGCTG-3’-MGBNFQ	SEQ ID No.6

Preferably, the concentration of the HCMV primer in the kit is 100nM, and the concentration of the probe in the kit is 100nM; the HBB primer concentration is 500nM, and the probe concentration is 200nM.

Further, in a preferred embodiment of the present invention, the kit further comprises a PCR reaction solution and/or a dye;

preferably, the PCR reaction solution comprises Taq enzyme, UNG enzyme, mg2+ buffer solution and dNTPs;

preferably, the dye comprises a ROX correction dye.

More preferably, in a preferred embodiment of the present invention, the amplification reaction system in the kit comprises:

HCMV reaction solution: a primer probe composition containing specificity aiming at a conserved region of an HCMV PP65 gene, and the sequence is shown as SEQ ID No. 1-3; internal reference primers and probes aiming at HBB conserved sequence specificity, the sequences are shown as SEQ ID No. 1-3, ROX correction dye;

HCMV premix a: taq enzyme, UNG enzyme;

HCMV premix B: PCR buffer solution, dNTPs and Mg ²⁺ Etc.;

positive control: pseudovirus containing HCMV target sequence, and plasmid containing human HBB gene target sequence.

Negative control: RNase-free ddH ₂ O。

In a third aspect, the present invention also provides a method of using the above kit for non-disease diagnosis and/or treatment purposes, comprising:

extracting the genome of a sample to be detected, amplifying the genome of the sample to be detected by using the kit, detecting a fluorescent signal, and analyzing.

Further, in a preferred embodiment of the present invention, the amplification procedure comprises:

further, in a preferred embodiment of the present invention, analyzing the fluorescent signal comprises:

when the amplification curve in the FAM fluorescence channel is S-shaped and Ct is less than or equal to 36, judging that the sample to be detected is positive to the human cytomegalovirus;

and when the Ct in the FAM fluorescence channel is greater than 36 or no amplification exists, the amplification curve in the VIC fluorescence channel is S-shaped, and the Ct is less than or equal to 36, the sample is judged to be negative to the human cytomegalovirus.

When Ct in the FAM fluorescence channel is greater than 36 or no amplification and Ct in the VIC fluorescence channel is greater than 36 or no amplification, the experiment is judged to be abnormal, and the sample needs to be extracted again for detection or the sampling needs to be performed again for detection.

Compared with the prior art, the invention at least has the following technical effects:

the kit realizes the detection of the nucleic acid of the human cytomegalovirus by using a Taqman probe and a real-time fluorescent signal under the action of taq DNA polymerase after extracting the nucleic acid from the collected sample by a conventional method. The kit does not have a complex nucleic acid extraction process, and the fluorescent quantitative PCR process is carried out by adopting a one-step method. The kit for detecting the human cytomegalovirus at least has the following beneficial effects:

1. the clinical diagnosis efficiency is accelerated: the invention provides a primer probe set, a kit and a using method for real-time fluorescence PCR detection of human cytomegalovirus, which can quickly, accurately and sensitively detect the human cytomegalovirus, and have good experimental result repeatability and high precision. The invention has short detection time period, can finish detection in 70 minutes at the fastest speed, greatly saves the detection time and accelerates the clinical diagnosis efficiency.

2. And (3) quality control in the whole process: the invention adds the monitoring of the reference gene, can monitor the quality of the whole process of sample extraction and amplification, can monitor whether DNA is successfully extracted and the PCR process is smoothly carried out, and can monitor whether manual misoperation occurs (the whole process is carried out in a closed tube state).

3. The operation is simple: the method can be used for detection on the computer only by mixing the human cytomegalovirus nucleic acid detection solution with the template by an operator, the detection instrument only depends on a fluorescent quantitative PCR instrument, the result Ct value instrument can be automatically interpreted, the requirement on the operator is extremely simple, and the clinical popularization is high.

Drawings

FIG. 1 shows the results of the positive control in example 2;

FIG. 2 shows the results of the detection of the negative control in example 2.

Detailed Description

Embodiments of the present invention will be described in detail with reference to the following examples, but those skilled in the art will understand that the following examples are merely illustrative of the present invention and should not be construed as limiting the scope of the present invention, and that the specific conditions not specified in the examples are carried out according to conventional conditions or conditions suggested by the manufacturer, and that the reagents or equipment used are not specified by the manufacturer, and are all conventional products available through commercial purchase.

The following describes in detail specific embodiments of the present invention. It should be understood that the detailed description and specific examples, while indicating the present invention, are given by way of illustration and explanation only, not limitation.

Example 1 development of fluorescent quantitative PCR kit

According to the report of the literature, the invention finally selects a primer probe designed aiming at the human cytomegalovirus gene region, selects the full length of the human cytomegalovirus pp65 gene in an NCBI database to carry out sequence comparison, designs the primer probe in the conserved region thereof, thereby ensuring that a pair of primers and one probe can detect all human cytomegalovirus strains, and ensures that the primer probe can only compare the human cytomegalovirus and can not compare the human cytomegalovirus strains with other pathogenic bacteria through BLSAT comparison.

Meanwhile, in this example, the human HBB gene was used as an internal reference gene, and specific primers and probes were designed.

a) The primers and probes for amplifying human cytomegalovirus HCMV (pp 65 gene conserved region) are shown as follows:

HCMV	nucleotide sequence	Serial number
			Forward sequence (qF)	5’-CCGGCAAGCTCTTTATGCA-3’	SEQ ID No.1
Reverse sequence (qR)	5’-TCATCGTCAGGTCCTCTTCCA-3’	SEQ ID No.2
			Fluorescent probe (qP)	5’-FAM-TCACGCTGGGCTCT-3’-MGBNFQ	SEQ ID No.3

b) The primers and probes for amplifying the internal reference HBB gene (conserved region) are shown in the following table:

the fluorescent quantitative PCR kit comprises:

(1) A primer probe set: the HCMV primer concentration is 100nM, and the probe concentration is 100nM; the HBB primer concentration is 500nM, and the probe concentration is 200nM.

(2) One-step amplification reaction solution: purchased from Nanjing Novowed Biotechnology Ltd (cat # Q222-CN), the reaction system comprises Taq enzyme, UNG enzyme, PCR buffer solution, dNTPs and various ions required by PCR amplification reaction solution.

(3) Positive reference (pseudovirus containing human cytomegalovirus target sequence, engineering bacterial strain of human HBB gene target sequence),

(4) Negative control (RNase-free ddH 2O).

The kit was stored at-20 ℃.

Example 2 fluorescent quantitative PCR detection method of human cytomegalovirus nucleic acid

This example provides a method for using the kit of example 1, namely a method for fluorescent quantitative PCR detection of human cytomegalovirus nucleic acid, comprising:

(1) Sample treatment: the required DNA can be obtained by taking 200 mu L of sample to be detected and extracting according to the conventional nucleic acid.

(2) Real-time fluorescent quantitative PCR amplification:

a. PCR amplified MIX (25. Mu.L per reaction) was prepared as in Table 1:

TABLE 1 PCR amplification reaction System

The prepared PCR-amplified MIX was dispensed in 25. Mu.L/reaction tube. Adding 5 mul of processed sample to be tested, positive reference substance and negative reference substance into corresponding reaction holes, and performing PCR amplification on the reaction holes.

b. And (3) amplification procedure:

in the parameter setting of the fluorescent quantitative PCR instrument, ROX is used as reference fluorescence. The present invention uses ABI 7500 for detection.

(3) And (4) analyzing results:

when the amplification curve in the FAM fluorescence channel is S-shaped and Ct is less than or equal to 36, the sample is judged to be positive for human cytomegalovirus;

when Ct >36 in FAM fluorescence channel or no amplification; when the amplification curve in the VIC fluorescence channel is S-shaped and Ct is less than or equal to 35, the sample is judged to be negative to human cytomegalovirus;

when the Ct in the FAM fluorescence channel is greater than 36 or no amplification exists, and the Ct in the VIC fluorescence channel is greater than 36 or no amplification exists, the experiment is judged to be abnormal, and the sample needs to be extracted again for detection or the sample needs to be sampled again for detection.

Wherein, the detection result of the positive control is shown in figure 1; the results of the negative control are shown in FIG. 2.

Example 3 performance assay of HCMV kits

1. And (3) measuring the accuracy:

taking 9 clinical positive vaginal swabs which are diagnosed as being infected by human cytomegalovirus as a sample to be detected, extracting total DNA, and adjusting the concentration to obtain a sample DNA solution to be detected;

preparing PCR amplification MIX according to the table 1, adding 5 mul of each of the sample DNA solution to be detected, the positive reference substance and the negative reference substance into corresponding reaction holes, and performing PCR amplification on the mixture by a computer.

The results are shown in table 2:

TABLE 2 test results of clinical positive samples

As can be seen from Table 2, the results of the 9 clinical positive samples tested by the kit are all positive, and the positive coincidence rate is 100%, thus showing that the kit has high detection accuracy.

2. And (3) specific determination:

clinical samples negative to 16 pathogenic bacteria such as mycoplasma pneumoniae, mycoplasma urealyticum, mycoplasma hominis, and chlamydia trachomatis were taken as test samples, and fluorescence PCR detection was performed using the kit provided in example 1 according to the method used in example 2.

The results are shown in Table 3:

TABLE 3 detection results of the kit on different pathogenic bacteria

In Table 3, UD indicates no Ct value or Ct >36.

As can be seen from the results in Table 3, the kit has strong specificity for detecting human cytomegalovirus, as only the detection result of human cytomegalovirus is positive and the detection results of other pathogenic bacteria are negative, by detecting various pathogenic bacteria.

3. Repeatability test

And (3) detecting by using 2 positive controls, repeating each sample for 10 times, and calculating the variation coefficient of the Ct value of the detection result.

Wherein, the calculation formula of the coefficient of variation (CV,%) is as follows:

in the formula:

CV-coefficient of variation;

s-standard deviation of Ct value;

-average of Ct values.

The results are shown in Table 4:

TABLE 4 results of repeatability measurements

As can be seen from Table 4, the coefficient of variation (CV%) of the Ct value in the results of the measurement was not more than 5% by repeating the measurement 10 times for each of 2 samples. Therefore, the kit has good repeatability on HCMV detection.

4. Minimum limit of detection

10 parts of the mixture with the concentration not higher than 1.0X 10 is used ⁴ copies/mL liquid indoor quality control containing human cytomegalovirus target sequence was used as a self-established positive control, and minimal detection limit detection was performed using the kit provided in example 1 according to the method of use in example 2.

The results are shown in Table 5:

TABLE 5 measurement results of detection limits

As can be seen from Table 5, the FAM signal channel Ct values of the detection results of 10 samples are all less than 36, and are all positive, and the lowest detection limit of the kit for HCMV detection is 1.0 multiplied by 10 ⁴ copies/mL, thus indicating that the detection sensitivity of the kit is high.

Finally, it should be noted that: the above description is only a preferred embodiment of the present invention, and is not intended to limit the scope of the present invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

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Claims

1. A primer probe composition for detecting human cytomegalovirus nucleic acid, which is characterized by comprising a real-time fluorescent PCR primer and a probe;

the real-time fluorescent PCR primer comprises a nucleotide sequence shown as SEQ ID No. 1-2;

the probe comprises a nucleotide sequence shown as SEQ ID No. 3.

2. The primer probe composition for detecting human cytomegalovirus nucleic acid of claim 1, wherein the probe is a Taqman probe, a fluorescent reporter group is labeled at the 5 'end of the Taqman probe, and a fluorescent quencher group is labeled at the 3' end of the Taqman probe.

3. The primer probe composition for detecting human cytomegalovirus nucleic acid of claim 2, wherein the fluorescent reporter group comprises at least one of FAM, cy5, ROX, VIC, and NED;

preferably, the fluorescent reporter group is FAM.

4. The primer probe composition for detecting human cytomegalovirus nucleic acid of claim 3, wherein the fluorescence quenching group comprises at least one of MGB-NFQ, QSY and BHQ 1;

preferably, the fluorescence quenching group is MGB-NFQ.

5. A kit for detecting human cytomegalovirus nucleic acid, comprising the primer probe composition of any one of claims 1 to 4.

6. The kit for detecting human cytomegalovirus nucleic acid of claim 5, further comprising an internal reference primer and an internal reference probe specific to human HBB gene conserved sequence;

preferably, the internal reference probe comprises the nucleotide sequence shown in SEQ ID No. 6.

7. The kit for detecting human cytomegalovirus nucleic acid of claim 5, further comprising PCR reaction solution and/or dye;

preferably, the PCR reaction solution comprises Taq enzyme, UNG enzyme, mg ²⁺ Buffer solution and dNTPs;

preferably, the dye comprises a ROX corrective dye.

8. A method of use of the kit according to claims 5 to 7 for the purpose of non-disease diagnosis and/or treatment, characterized in that it comprises:

9. The use of claim 8, wherein analyzing the fluorescent signal comprises: